Immunoassay apparatus for diagnosis

ABSTRACT

Immunoassay analytical test apparatus for allergy diagnosis, which apparatus comprises a zone for receiving a sample containing an analyte, a zone for receiving a mobile phase (the zone may be the same as the sample receiving zone, or different thereto), a detection means for permitting detection of the analyte by immunoreaction, a first flow path for flow of the analyte in the mobile phase from the sample receiving zone to the detection means, and a second flow path permitting flow of a mobile phase to the detection means.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is a continuation in part ofinternational application PCT/GB98/01412 filed on 15th May 1998 by thesame applicant as the present invention.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to apparatus for the diagnosis ofallergies. In particular, the invention relates to immunoassay apparatusthat does not require sophisticated laboratory equipment or technicalexpertise and is suitable for use in the home or the doctor's surgery.

[0003] A number of kits using immunoassay technology (mainly forpregnancy testing or fertility prediction) are widely available for usein the home or in a doctor's surgery. The format of the immunoassaytechnique in such kits is broadly similar, utilizing test stripscontaining immobilized immunoreactant and requiring the user to provideand apply a sample of a predetermined body fluid. Some devices that useurine as the source of the analyte require no further intervention thanthe application of the sample to the device. This is an ideal situationwhere urine not only contains the analyte but also acts as the fluidcomponent of a mobile phase which initiates the chemical reaction withinthe device.

[0004] However, other samples may not be readily available in suchcopious volumes. For applications where urine is not appropriate, ablood sample is usually required, although other body fluids may also beof use, for example, saliva or tears. Due to practical and ethicalreasons, devices that are designed for use in the home and that requirea blood sample must work with a capillary sample not usually larger thana few hundred microliters in volume.

[0005] For most devices, because of physical constraints such as flowproperties, this volume is insufficient to initiate the chemicalreaction and permit it to proceed. Additional fluid components must thenbe added to the kit in order to provide a mobile phase. This mobilephase can be contained within a compartment inside the apparatus, therelease of which is initiated either by physical intervention by theuser, or by a chemical interaction of the sample with a physical barrierseparating the mobile phase from the immunoreactants. The mobile phasecould also be added from a separate container to a receptacle on thedevice itself. Of course the more steps that are involved with theoperation of the kit, the more chance there is for error by the user andless is the likelihood of obtaining a correct result.

[0006] For some medical conditions, it is important to obtain a rapidtest result. For example, a result within minutes of sample applicationmay be required with a test to be used in emergency situations, whereasfor other conditions a result within minutes may not be necessary.

[0007] Currently available apparatus is often non-sequential in nature,meaning that the reaction involves a single step. However, one of themain problems with non-sequential immunometric test kits is theoccurrence of a phenomenon called a “high dose hook effect”. The highdose hook effect is evident when high levels of antigen in the systemsaturate the assay. This is caused by free analyte being left in thesample after reaction of all the available labelled immunoreactant andthe inadequate binding capacity of the immunoadsorbent for the amount ofcomplexed and free analyte in the system. This unlabelled analyte canthen compete downstream for the immobilised immunoreactant. In somecases, when there is a large amount of analyte in the sample, falsenegative results can be obtained merely because of saturation of thelabelled immunoreactant and immunoadsorbent. These false negative, orabnormally low, results may influence decisions on treatment made by theuser or clinician. This will not occur if sufficient immunoadsorbent ispresent. However, there are limitations to the binding capacity of animmunoadsorbent, and it is not generally practical to increase theamount of labelled immunoreactant in the system to overcome the highdose hook effect, as this will tend to increase the non-specific bindingof the labelled immunoreactant, thereby reducing the analyticalsensitivity of the assay, giving rise to an elevated reference (blank)reading. In conventional laboratory immunoassays, this problem isovercome by carrying out a sequential assay, with discrete wash stepsbetween each addition of sample and immunoreactant. Sequentialimmunometric assays can be easily run in a laboratory. For example, asample which contains analyte may be allowed to react with animmobilized immunoreactant specific for the analyte in question. After apredetermined incubation period, unbound analyte in the sample may bewashed away, usually by a combination of decantation and washing. Thenthe labelled immunoreactants are added.

[0008] A further method to circumvent the high dose hook effect, and toavoid the sequential format, would be to use a competitive assay format.However, this is not ideal. For example, with such assays, the precisionis highly dependent on the region of the dose response curve beingexamined. In contrast to immunometric assays, the response incompetitive assays is inversely proportional to the dose—that is, alower signal is obtained with higher concentrations of analyte. Ingeneral, this is not a preferred attribute for a home testing kit wherethe absence of a signal confers a positive result. A preferred way toavoid the high dose hook effect while maintaining an acceptable assayformat is a sequential immunometric assay approach. However, therequirement for separate addition of reagents by the user andintervening discrete wash steps, would make such a testing device toocumbersome for home or office use.

[0009] EP 0314499 A discloses a test device for carrying out animmunoassay and making a color change result visible to the user. Thedevice comprises a first “fast” flow path and a second “slow” flow pathof porous or fibrous liquid conductive material. The ends of both tracksare contacted with a sample liquid store (mobile phase containing theanalyte). The first path for flow of the analyte contains an enzyme andthe second path contains a labelling substrate corresponding to theenzyme. Both paths lead to a detection means.

[0010] EP 0186799 A discloses an analytical device comprising a zone forreceiving sample containing an analyte, a zone for receiving the mobilephase and a detection zone. The document teaches the possibility ofhaving two parallel flow paths which start from the mobile phasereservoir and lead the detection zone, wherein the main path transportsthe sample containing the analyte and the second path entrains reagentsfor color detection.

[0011] EP 0590695 A discloses a liquid transfer device for use in assayprocedures, comprising a sheet of porous material for capillary liquidflow therethrough. The sheet is formed to define two liquid flowchannels leading from two channel ends to a site in a common channelportion, where the two channels merge. The two channels are operable todeliver liquid to said common site in a sequentially timed mannerfollowing simultaneous application of such liquid to the channel ends.

OBJECTS OF THE INVENTION

[0012] The present invention aims to overcome the problems associatedwith non-sequential immunometric assays by providing a self diagnosisapparatus which can use a sequential immunoassay method, but which doesnot require sophisticated laboratory equipment or technical expertise.

[0013] It is an object of the present invention to provide a selfdiagnosis apparatus utilising immunoassay technology which isparticularly useful for the measurement of antigen-specific humanimmunoglobulin E (IgE), as in allergy diagnosis, but which may also beused for other analytes and isotypes.

SUMMARY OF THE INVENTION

[0014] Immunoassay analytical test apparatus for allergy diagnosisaccording to the present invention comprises:

[0015] (a) a zone for receiving a sample containing an analyte;

[0016] (b) a zone for receiving a mobile phase, which zone may be thesame as the sample receiving zone, or different thereto;

[0017] (c) a detection means for permitting detection of said analyte byimmunoreaction;

[0018] (d) a first flow path for flow of said analyte in said mobilephase from said sample receiving zone to said detection means; and

[0019] (e) a second flow path permitting flow of said mobile phase tosaid detection means.

[0020] It is a preferred feature of the present invention that thesecond flow path includes therein labelled immunoreactive materialcapable of reacting with the analyte to produce labelled anylate.

[0021] It is a preferred feature of the present invention that the firstflow path includes unlabelled or hapten labelled (such as biotinylated)immunoreactive material being placed upstream and/or downstream of thesample receiving zone. It is a further preferred feature that the firstflow path includes both unlabelled or hapten labelled capture materialand detector labelled material. The materials may be dried onto the flowpath substrate upstream and/or downstream of the sample receiving zone.As the mobile phase containing the analyte flows towards the detectionmeans the dried material may be solubilized by the mobile phase.

[0022] The problems associated with non-sequential immunometric assaysmay be alleviated by permitting the analyte to reach the detectionmeans, where it is immunoextracted by an immunoadsorbent, before asubstantial amount of labelled immunoreactive material reaches thedetection means.

[0023] According to a first embodiment of the present invention this maybe achieved by providing detection means which may be separable from theflow paths, thereby allowing the detection means to be moved from theflow path comprising the analyte to the flow path comprising labelledimmunoreactive material only after sufficient immunoadsorption of theanalyte in the detection zone has taken place.

[0024] Therefore, according to a first embodiment of the presentinvention the test apparatus comprises:

[0025] (a) a zone for receiving a sample containing an analyte;

[0026] (b) a zone for receiving a mobile phase;

[0027] (c) a first flow path for flow of an analyte in said mobile phasefrom a sample receiving zone to a detection means;

[0028] (d) a second flow path for flow of a mobile phase from a mobilephase receiving zone to detection means, said flow path having therein azone containing labelled immunoreactive material capable of reactingwith said analyte to produce labelled analyte; and

[0029] wherein the detection means is movable from a first position incommunication with said first flow path to a second position incommunication with said second flow path.

[0030] It is a preferred feature of the first embodiment of the presentinvention that the detection means is movable to be in communicationwith the first and second flow paths in sequence. It is furtherpreferred that there is contact between the detection means and thefirst and second flow paths.

[0031] The flow paths according to the present invention compriseelongate sheet or strip material having a portion connectable with saidfirst and/or said second flow path. The flow paths are designed topotentiate flow toward the detection means by permitting movement ofmobile phase in a longitudinal direction.

[0032] According to a second embodiment of the present invention thearrival of the labelled immunoreactive material to the detection meansmay be delayed by having a longer flow path for the labelledimmunoreactive material relative to the flow path for the analyte.

[0033] Therefore, according to a second embodiment of the presentinvention, the test apparatus comprises:

[0034] (a) a zone for receiving a sample containing an analyte;

[0035] (b) a zone for receiving a mobile phase, which zone may be thesame as the sample receiving zone, or different thereto;

[0036] (c) a detection means for permitting detection of said analyte byimmunoreaction; and

[0037] (d) first and second flow paths for flow of said analyte in saidmobile phase from said sample receiving zone to said detection means,said second flow path having therein a zone containing labelledimmunoreactive material capable of reacting with said analyte to producelabelled analyte, such that unlabelled analyte reaches said detectionzone via said first path before said labelled immunoreactive materialreaches said detection zone via said second path whereby said labelledimmunoreactive material can react with immobilized analyte.

[0038] According to the second embodiment of the invention, it ispreferred that the flow paths are such that the mobile phase is allowedto traverse more than one route simultaneously. The first and secondflow paths are preferably both continuous from the mobile phasereceiving zone to the detection means. The most direct route of themobile phase is through the sample receiving zone and into theimmunoadsorbent. However, simultaneously, the mobile phase can alsotravel along a different conducting element. Within this alternate routethe mobile phase passes through a zone of labelled immunoreactant whichis free to move in the conducting element when in the moist state only.This alternate path of mobile phase carrying the labelledimmunoreactants also leads to the immunoadsorbent.

[0039] The immunoassay apparatus according to the present inventionpreferably comprises an external body and an internal body. The externalbody typically includes a receptacle for the addition of a samplecontaining the analyte, and a compartment where the result of the testcan be observed.

[0040] Furthermore, the external body of the apparatus may comprise alancet to enable the user of the device to provide a blood sample. Thelancet may be integrally molded to the external body of the apparatus,or it may be provided separately.

[0041] Furthermore, a desiccant tablet may also be included in theapparatus and the entire unit hermetically sealed to preventdeterioration through humidity changes. The apparatus is particularlysuited for use in the home, at a patient's bedside or in a doctor'soffice.

[0042] A further embodiment of the device is the inclusion of a recessinto the external body of the apparatus to which a drop of blood isadded. It is preferred that this recess leads directly to the surface ofthe first flow path. The recess may be arranged to prevent the usertouching the surface of the flow path during the course of the test.

[0043] All the components necessary to obtain a result, except for thesample containing the analyte to be detected, may be provided as a kitand therefore the use of the apparatus according to the invention is notlimited to the laboratory.

[0044] To operate the apparatus according to the present invention, auser provides a sample containing the analyte, for example, a sample ofblood. A capillary blood sample can be taken using a lancet (which ispreferably incorporated into the apparatus) to prick a finger. The bloodor other sample is then allowed to be taken up by a sample receivingzone. The sample receiving zone preferably comprises a suitable bloodfilter to aid separation of plasma from the whole blood sample. Mobilephase may then be applied upstream of the sample.

[0045] Aliquots of mobile phase may be applied indirectly to the mobilephase receiving zone from a separate container holding mobile phase, forexample, from one or more dropper bottles. The mobile phase, in thiscase, would be applied onto an adsorbent solid phase which is acontinuation of the body of the testing apparatus, or it may be appliedin the form of a well with a connecting chamber to the sampleapplication zone and other conducting elements. Alternately, the mobilephase, which typically comprises physiological saline and antimicrobialpreservatives, may be released into the apparatus directly from anintegrated reservoir. In this case, the mobile phase may be containedwithin a blister or the like, permitting release of the mobile phaseduring operation of the apparatus. The mobile phase may be released byapplying pressure or, for example, by suitable piercing means, to thesurface of the blister causing the blister to be punctured. The piercingmeans may be incorporated into the apparatus. Once punctured, pressureon the top of the blister causes deformation of the blister therebysqueezing out the mobile phase from the blister. Alternately, the directrelease of mobile phase may be by chemical intervention. The chemicalintervention may be initiated by compounds in the sample acting to“digest” a membrane, thereby releasing the contents of a reservoir forthe mobile phase. No further intervention is required by the user exceptto read the result of the test, a positive result being indicated bycolor in the detection zone, after a predetermined incubation time.

[0046] As previously mentioned, the present invention is particularlysuitable for use at home or in the doctor's surgery. It is thereforeimpractical to obtain a blood sample greater than a few hundred μl involume, a preferred volume being less than 50 μl. This volume of wholeblood is roughly 50% cellular which normally leaves less than 20 μl ofliquid sample available for analysis. Allergy tests have to be highlysensitive to detect minute amounts of IgE present in the circulatingblood. It is therefore advantageous to extract as much of the plasmainto the test system as possible. A system that uses the sample as asource of mobile phase for the labelled immunoreactant and subsequentwash steps of a sequential immunoassay is therefore impractical for theminute volumes available in this instance. Here a separate mobile phaseis added from a reservoir that pushes the plasma out of the samplelaterally through the filter.

[0047] Advantageously, it is this lateral separation rather thantransverse flow of the capillary blood sample through a suitablefiltration means (which may advantageously comprise the material of thefirst flowpath) which results in the efficient extraction of plasmacomponents free of red cells. The mobile phase pushes the plasmacomponent out of the sample causing the plasma to travel laterally alongthe length of the flow path (rather than causing separation through thedepth of a filter in a conventional manner which would cause asignificant fraction of the plasma to be retained). The sample, free ofcells, is drawn laterally through the flow path by the addition of amobile phase, which may also contain labelled reactants, towards theimmunoadsorbent. This is in contrast to single step devices in which theimmobilized reactants nearest to the application zone are exposed tomore of the analyte than immobilized reactants more distal to theapplication zone.

[0048] In a preferred embodiment of the present invention, the apparatusadditionally comprises a blood filter. A sample of whole blood(typically approximately 50 μl) may be applied to the surface of asuitable cell filter, for example, a CytoSep filter (Ahlstromfiltration) or a glass fiber filter such as GF/A (Whatman), or GF51(Schleicher and Schuell) which may be pre-treated with detergents,anticoagulants and other reagents common in immunoassay technology. Thereceptacle housing such a filter protects the user from the chemical andallergenic contents of the apparatus. The filter may contain a chemicalanticoagulant such as oxalate or fluoride, a chelating agent (such asEDTA) or an anticoagulant such as heparin. Some cells may also beremoved by specific binding agents immobilized close to the sampleapplication zone, such as polyfunctional group reactive lectins. Thecells could also be lysed by physical or chemical shock, such as bychange in the pH or ionic environment, freezing, heating, desiccation orthe action of specific biological lysing agents or organic compounds.However, for some analytes, removal of the cells will not be necessaryfor the successful use of the apparatus according to the invention.

[0049] Once a sample of blood or the like has been fully adsorbed, theplasma may be separated from the cells by allowing the free end of thefilter to come into contact with a solution containing anti-human IgE,which may be hapten or directly labelled, together with preservativesand stabilizers (common to the practice of immunoassay development usedto minimise non-specific interactions). However, the use of ananti-human IgG may be useful when diagnosing food allergies that are notdependent on IgE mechanisms. If an indirect label is added at the abovestep, then a second reagent reactive with labelled groups on theanti-IgE or anti-IgG antibody can be added by simply exposing the freeend of the filter to a reagent containing the labelled second antibody.The latter method thereby increases the sensitivity of the detectionmethod. After a predetermined time, the immunoadsorbent will display afingerprint of the allergic profile of the patient as a series of bandswhich can be attributed to the existence of allergen specific IgE or IgGantibodies, hence suggesting the possibility of IgE/IgG mediatedallergic reactions against those allergens tested.

[0050] Referring specifically to the embodiment described with movabledetection means, the mobile phase may be absorbed simultaneously alongtwo separate paths leading from the mobile phase receiving zone. Thesample receiving zone may comprise a blood filter. This will allowlateral separation of the plasma from the cells and subsequent flow ofthe plasma into the detection means. After a fixed time, typically 10minutes, the plasma component of the blood sample will have beenextracted from the blood sample and passed through to the detectionmeans, followed by a mobile phase which has the effect of removing freeunreacted analyte in the plasma from the detection means.

[0051] The blood filter may additionally contains additives for thespecific removal of non-IgE isotypes from the plasma. As the plasmasample from a positively testing subject may contain antibodies directedagainst the allergens in the detector zone, other than allergen specificIgE (which condition may be quite marked in patients with foodallergies) there will be a tendency for the IgG antibodies, for example,in the plasma sample, to swamp out the binding of the IgE antibodiesagainst the allergen. This could result in a false negative result beingreported for the plasma sample due to inhibition of the test by non-IgEantibody isotypes. Here much of the plasma, and hence analyte, passesthrough the blood filter that aids removal of the non-IgE antibodies.The means for removal of non-IgE components may comprise ion-exchangematerial that allows IgE antibodies to pass through unaffected but bindsto the non-IgE isotypes preventing their entry into the detection means.

[0052] The matrix or filter for the removal of non-IgE components may beprovided separate from the blood filter. The blood filter and/or thematrix for removal of non-IgE components are preferably located betweenthe sample application zone and detection means.

[0053] An important embodiment of the apparatus according to theinvention which increases the specificity of the diagnostic test is thatthe sample, for example for measuring allergen specific IgE, may beadsorbed free of non-IgE isotypes by passing the sample through a matrixcontaining an adsorbent reactive with human IgG, IgM or IgA e.g.lectins, protein A or anti-human antibodies, other than the specificantibodies used, thereby facilitating the adsorption of non-specificisotypes (for measurements of IgE, IgD, and IgA or IgG subclasses).Incorporation of an adsorption matrix between the sample applicationzone and the immunoadsorbent will yield more specific and sensitiveallergy assays by excluding interference with non-IgE antibodies.Alternately, the matrix may be provided in the sample application zonewithin the blood filter. Alternately, the matrix is provided separate tothe blood filter. It is also possible to immobilize antibodies toprovide a zone in the detection means prior to the allergen impregnatedmeans. For example, the presence of large quantities of non-IgEanti-allergen antibodies in the sample will inhibit the binding of IgEantibodies by competition for the allergosorbent, giving the possibilityof a false negative result which may be particularly important for thediagnosis of food allergy mediated by IgE, or where patients haveundergone immunotherapy with allergen extracts (which may result in theelevation of non IgE immunoglobulin isotypes). It would therefore beexpected that an assay using the apparatus according to the inventionwould classify some patients as reacting positively to certainallergens, while other commercial allergy assays may have classified asnegative or as a lower allergy class. This may be an important featurewhen objective measurements of IgE in the absence of other isotypes aredesired, or when specific isotypes of antibody are measured withoutinterference from other isotypes. A further implication of thisembodiment is the removal of immune complexes of allergen, IgE and IgGbecause of the polyclonal nature of the immune response to a number ofepitopes on the allergen surface. Removal of such complexes coulddecrease the number of asymptomatic specific IgE positive results, whichis currently found with current classical allergy tests for themeasurement of specific IgE. The absorption technique could also beextended to the removal of crossreactive antigens in the case ofimmunoassays for other analytes, or for the removal of interferingcomponents (such as immune complexes as in certain disease conditions)and also where biologically active components or chemically interferingmaterials (such as proteases and other binding substances) need to beremoved.

[0054] The mobile phase also simultaneously passes along the second flowpath through the labelled immunoreactive material to the detection zone.During the initial ten minute separation of the plasma from the wholeblood sample, the mobile phase also serves to rehydrate dried developerreagent, such as a labelled anti-human IgE antibody, in the second flowpath.

[0055] After a specified period of time, typically ten minutes, thesecond phase of reaction is initiated by the physical movement of thedetection zone from the first flow path to the second flow path. Thisprocess may be carried out manually or by other means. Now the activeflow path is from the reservoir or mobile phase receiving zone throughthe rehydrated labelled antibodies into the detection zone. An importantfeature of the breaking of the flow of the first flow path is that thereis little chance for the red cells from the blood sample to diffuse intothe detection zone and thereby obscure the result on the detectionfilter.

[0056] The labelled immunoreactants pass through the detection zone as abolus which serves two purposes:

[0057] (i) the concentration of the labelled immunoreactants in thedetection means is increased hence increasing the reaction rate; and

[0058] (ii) the bolus is followed by a zone of mobile phase which servesto clear the detection zone of remaining labelled immunoreactantsthereby reducing any nonspecific binding and increasing the signal tobackground readings.

[0059] At the end of the detection means lies a sink which may or maynot be always in contact with the detection means except during thesecond phase of the test whereby the detection means and the second flowpath are in contact. This area serves as a sink for unreacted materialand can thereby accumulate unreacted (excess) labelled immunoreactantsthereby changing the visual appearance of the sink either directly inthe case of particulate labels or indirectly through reaction of thelabelled immunoreactants by a further developer. This enables the excesslabelled immunoreactants to serve as a means of determining when thetest is complete.

[0060] The movement of the detection means is a key feature. Thedetection means is clipped into the second flow path and importantlyholds the filter at an angle to the surface of the detection means. Thefirst flow path and second flow path are also inclined at a similarangle such as when the detection means is pulled from the first flowpath, the detection means is forced against the second flow path,ensuring a good contact to allow continuity of the flow of the mobilephase and reactants it carries. The detection means may also contain awindow through which the result of the test can be observed.

[0061] According to another feature of the present invention, anallergosorbent may be used. Due to the high sensitivity required for thedetection of small amounts of allergen specific IgE compared to allergenspecific IgG, and the fact that the IgE may be directed to only a smallfraction of the allergosorbent, the properties of the substrate forbinding the allergens should be highly efficient. The flow rate is alsoimportant, because too rapid a flow rate would result in too low asensitivity. A plastics backed nitrocellulose membrane (SSLU, Schleicherand Schuell) with a pore size of 3 to 5 μm, or a Whatman 3 mm, has beenfound to be suitable for the determination of allergen specific IgE.

[0062] The apparatus according to the invention may be used for thedetection of multiple allergen specific IgE rather than IgE towards asingle allergen. For example, in the case of the embodiment according tothe present invention with non-movable detection means, the rate of flowthrough the allergosorbent will be proportional to the length of therespective flow path. Hence the larger the panel of allergens tested,the longer will be the development time for a result to be observed.

[0063] The allergens may be applied to the nitrocellulose with a pencapable of dispensing aqueous allergen extracts at 1 μl per linear cm atconcentrations ranging between 1 to 10 mg/ml in 50 mM Tris bufferedsaline pH7.4 (the protein concentration depending on the source ofextract used). The nitrocellulose may then be allowed to dry at roomtemperature for 30 minutes. The excess reactive groups on thenitrocellulose may then be blocked for 1 hour with a Tris bufferedsaline solution containing 0.05% v/v Tween-20. A further wash with theabove buffer may be performed for 15 minutes prior to allowing themembrane to dry at room temperature for 3 hours and then storing thedesiccated material at 2 to 8° C.

[0064] It is preferred that the detection means comprises animmunoadsorbent. Many current immunoassay devices operate by the sampleflowing through a zone of labelled immunoreactants downstream from thesample application zone, which immunoreactants bind specifically withthe analyte if found to be present in the sample. The label constitutesthe means by which the specific analyte is made detectable to the user.This labelled immunoreactant can be direct, for example, when bound todyed latex particles, gold colloids or dye sols. The labelledimmunoreactant may also be indirect, such as when the label is abiological enzyme that requires treatment with a substrate and chromogenprior to detection of the label, or when silver enhancer reagents arerequired before previously undetectable gold labels are renderedvisible. The labelled immunoreactant may be free to move along with themobile phase. The labelled immunoreactant-analyte complex, if present,moves downstream according to the flow initiated by the reservoir ofmobile phase, towards an immunoadsorbent layer.

[0065] Here, for example, a further immunoreactant, again specific forthe analyte (but to a different reactive group on the analyte) can bindto the labelled immunoreactant-analyte complex, downstream from thelabelling zone. The presence of analyte in the sample can therefore bevisualized by the accumulation of labelled complex at the site of theimmunoadsorbent. In a further feature of the apparatus, depending on thenature of the labelled immunoreactants, the mobile phase may bewater-based (e.g. a saline solution), it may contain a substrate and/orchromogen for an enzyme label, or it may contain a silver enhancerreagent (such as one commercially available suitable for thevisualization of histological sections with colloidal gold reagents fromSigma-Aldrich Company) or one of the components of such a reagent whengold-labelled immunoreactants are used for detection (or indeed otherdetection agents).

[0066] The assay can be rendered more sensitive by the lateral flow of asilver enhancer solution. This allows the use of smaller gold colloidsgiving better penetration and resolution of the membrane but thenenhanced by the deposition of metallic silver on the surface of thecolloidal gold. This should be useful to the clinical laboratory whereapplications involving minute sample volumes are required or whereultra-sensitive detection systems are required for the simultaneousdetermination of multiple analytes from a minute sample. The goldconjugates may also be silver-coated prior to their inclusion within theapparatus according to the invention.

[0067] Such conjugates may be prepared by incubating a titered aliquotof gold labelled antibody (dialysed or suitably diluted to remove ionssuch as chloride), preferably 5 nm or less, with a reactive solution ofsilver enhancer for 5 to 10 minutes, followed by centrifugation andthree washes with equal volumes of distilled water, to remove excesssilver enhancer.

[0068] A positive result in such immunoassays using such pre-silverstained gold conjugates with silver-stained gold conjugate isrepresented by an intense black coloration at the binding site, therebyproviding sensitivities many orders of magnitude greater than that ofthe original gold colloid. These silver-stained gold conjugates can bereadily prepared and are stable in both liquid and dried form. Smallergold conjugates of 5 nm or less are most appropriate for silverstaining. The intensity of silver staining can be modulated by theconcentration of reactants, removal of interfering salts, temperature (areduction in temperature can be used to control the rate of reaction)and the type of labelling system used. These silver stained goldconjugates may also be of use where a passive immunoassay format isused, such as in classical protein and nucleic acid blotting.

[0069] In a further embodiment of the invention, there may be includedan onboard control that indicates when the assay has been complete orsuccessful. This control (or a further control) can also be used as areference to the test result obtained with the specific analyte. Thus,by comparing the intensity of the reference with the test result, anindication as to whether the test result was positive or negative willbe obtained. Hence, by means of a further apparatus that measures theintensity of the response, a quantitative measure could be achieved bycomparing the activity of the reference with the test result.

[0070] Alternately, the device may also contain an electronic means ofdetecting the result, the termination of the test and audible or visiblestep-by-step instructions for performing the test. Inclusion of theseelectronic features would allow greater confidence of the user inperforming the test correctly and reduce the requirement for the readingof instruction leaflets prior to use. For example, regions of the devicecould contain sensors that detect moistness of the flow paths thusdetermining the step the test is at during the processing of the sample.Such sensors could be linked to visual or audible displays of theprocessing step and indicate the next step. Such sensors in thedetection zone could include densitometric measurements, that determinethe amount of labelled antibody specifically bound by analyte in thedetection zone in order to produce a quantitative result or a means ofindicating when the detection means should be pulled to the developerposition such as an audible warning or visual instruction or signal. Theelectronic sensor could also indicate when sufficient blood has beenadded to the sample receiving zone by, for example, the determination ofthe change in conductivity of the filters between the dry and wet stateor when the ionic composition of the filter changes due to the additionof polar solvents or sample or by measuring a change in optical densitysuch as that resulting from the addition of a colored sample (as is thecase with a blood drop) by means of a photocell for example.

[0071] A further embodiment could include means for measuring the amountof sample added. This again could be done through electronic means asdescribed above or more simply through the use of an integral capillarytube or similar that would “draw up” a fixed volume of sample into thedevice for the analysis.

[0072] The detector could also take the form of an electronic devicewhere the physical properties of the solid phase to which the allergensare absorbed is changed by the binding of the labelled anti-human IgEantibodies to these zones when in the presence of the analyte ieallergen specific IgE.

[0073] The present invention therefore helps to eliminate commonpitfalls in immunoassay technology associated with known apparatus.

[0074] Sequential assay also allows amplification of the assay system bythe use of multiple additions of antibodies e.g. in the case of asensitive allergy test, the first antibody may be a biotinylatedanti-human IgE which is washed through by a gold-labelled anti-biotinsecond antibody which is readily visible to the naked eye whenconcentrated, in the form of a discrete reaction zone at theimmunoadsorbent site.

[0075] The intermission of discrete wash steps common to routinesequential assays as performed in clinical laboratories is unnecessaryfor this format of the assay, because the capillary nature of thefilters and membranes, as the first antibody is washed out with littlereaction and mixing by the second antibody. The same can be said aboutthe sample which is pushed out of the filter by the influx of theprimary antibody.

[0076] A principal embodiment of the apparatus according to theinvention is the circumvention of the requirement for discrete washsteps while still retaining a sequential immunometric assay format andis especially applicable for apparatus to be used in the home ordoctor's office. This is achieved by the construction and, in the caseof the embodiment with non-movable detection means, geometry of theconducting elements transporting the mobile phase. In contrast to knownapparatus, it is a feature of the apparatus according to the inventionthat the sample can be applied to a region close to the immunoadsorbent.Mobile phase can then be applied upstream of the sample either byrelease from a compartment within the device or added from a separatecontainer supplied as part of the apparatus. This circumvents thenecessity for the mobile phase to enter the labelled immunoreactant zonebefore reaching the sample.

[0077] Therefore, due to a possible difference in flow characteristicsof the two paths mentioned above, it is evident that arrival of thelabelled immunoreactants much later at the immunoadsorbent than theinitial analyte from the sample application point will effectivelyresult in a sequential immunometric assay. This procedure allows thesequential format described, to take place without the requirement of aprevious interaction with a labelled immunoreactant and without discretewash steps or separate multiple applications of reagents by the user.

[0078] In a further embodiment of the invention, the number of pathsneed not be limited to two. A further increase in the number of paths ofvarying flow characteristics provides more flexibility with the testingsystem, allowing more complex chemistries to take place or facilitateenhanced washing steps if required, or chemical modifications steps orreaction with different labelled immunoreactants in turn on the solidphase. Nevertheless, the only requirement for the user, irrespective ofthe number of flow paths, is to provide a sample at the applicationpoint and then to initiate the flow of the mobile phase either fromwithin the device or via an external container.

[0079] The most rapid path need not necessarily lead to the sample. Thepresent invention could also be applicable in carrying out theimmobilization of immunoreactants during operation of the apparatus,rather than immobilized immunoreactants being prepared by themanufacturer.

[0080] A sequential format is particularly useful when monoclonalantibodies are unavailable. In general, polyclonal antibodies canpotentially give greater sensitivity in immunoassays and can be morestable when labelled than monoclonal antibodies. It is important whenusing a non-sequential assay device, as described previously, that thelabelled antibody is specific for one epitope while the immobilizedimmunoreagent is specific for a spatially distinct distant epitope.However, in some cases this would preclude the use of polyclonalantibodies because of the possibility of reaction with the epitopesrecognized by the immobilized immunoreactant on the immunoadsorbent,thereby effectively masking the epitopes to the immunoadsorbent,reducing binding and hence reducing the signal.

[0081] A sequential assay format eliminates these problems and isparticularly useful when the analyte is an immunoglobulin or when onlypolyclonal antisera are available. The immunoadsorbent is usuallyimmobilized allergen extract. However, specific allergen assays may alsobe conducted by using an immobilized anti-human IgE antibody andlabelled allergens. The multiple simultaneous detection of IgE and IgGantibodies directed against an allergen could also be performed byadapting this method using labelled allergens. In this method, forexample, the detector may comprise two zones:

[0082] (a) a zone containing an immobilized anti-IgE antibody; and

[0083] (b) an immobilized IgG binding reagent such as protein A oranti-IgG antibody.

[0084] Unlike many immunometric assays where the immunoadsorbent is apreparation of purified antibody, for allergen extracts they are usuallycomplex mixtures of a multitude of proteins. For example, house-dustmite extracts contain many antigens and allergens each reactingdifferentially with blood samples from different individuals. Here,previous reaction of the IgE present in a capillary blood sample withlabelled anti-human IgE antibodies, as would occur using conventionaldevices for home/office testing, can possibly inhibit interactions withthe allergosorbent solid phase due to the orientation of theanti-IgE/IgE complex interfering with the binding of such complex to theimmunoadsorbent. A particular feature of most laboratory based allergytests is that they adopt a sequential assay format, thus circumventingthis problem of steric hindrance by allowing the IgE in the patientsample to bind to the allergen solid phase first. Then, a polyclonallabelled anti-human IgE antibody preparation can be used in excess toensure quantitative estimation of the specific IgE bound.

[0085] A major advantage of the use of the sequential format in adiagnostic device is in the case of celiac disease. Here the presence ofImmunoglobulin A (IgA) antibodies against the cereal protein gliadin inthe blood and some other body fluids, for example saliva, is anindication of celiac disease. However, the fraction of gliadin specificIgA compared to total IgA in blood samples from individuals with celiacdisease can be very small. This means that in order to detect gliadinspecific IgA, a sensitive detection system is required together with alarge sample volume, if the signal obtained with a sample containinggliadin specific IgA antibodies is to be distinguished from bloodsamples that are not. The increase in the sample size, corresponds to anincrease of total IgA respectively, which has the result that existingnon-sequential assays could be too insensitive, as there is apossibility that due to the increased sample volume and total IgAlevels, that the high levels of non-gliadin specific IgA couldpotentially swamp out the detection of the gliadin specific IgA bysaturation of the labelled anti-human IgA antibodies.

[0086] Thus the use of a sequential assay, of first allowing the humananti-gliadin specific IgA to bind to immobilized gliadinimmunoadsorbent, then reacting in a separate stage thereafter with thelabelled anti-human IgA, will result in a more specific, sensitive andreduced possibility of false negative assays which may have been causedby saturation of the assay system with the non-gliadin specific IgAfound in the blood sample. It is also possible that the mobile phasecould be used to cause a slow or gradual release of labelled reactantsfrom a matrix which would act in concert with the sequential assayformat described above to provide an even clearer separation ofimmunoreactants during operation of the device. For example, in the caseof specific IgA against gliadin, the assay would be relativelyinsensitive if non-specific IgA was not removed prior to detection withan anti-IgA label, which in conventional laboratory based immunoassaysis circumvented by a sequential assay format which incorporates a washstep to remove non-specific immunoreactants prior to the addition oflabelled antibody. However in a diagnostic apparatus designed for use inthe home or the office, discrete wash steps are inconvenient andcumbersome and a possible source of error.

[0087] The result depends on a number of factors including; the manualintensity of washing performed by the operator, the reagents used forthe wash step may differ, especially if tap water is advised e.g.contaminants, microbial, particulates, force and temperature.

[0088] To circumvent the requirement for a wash step, the apparatusshould contain where necessary means for the removal of non-specificreactants. It is therefore a further embodiment of the apparatusdescribed here that, if necessary and depending on the analyte to bemeasured, means for the slow release or desorption of the labelledimmunoreactants should be an integral part of the operation. Inpractice, such a slow release mechanism could take the form of amechanical barrier, for example, a gel or encapsulating material, orchemical means such as adsorption to a charged matrix such as anion-exchange material or affinity adsorbed by a specific property ofbinding of the labelled reactant for an immobilized ligand and desorbedby a suitable change in ionic strength, hydrogen ion concentration, oraddition of compounds that antagonize ligand-reactant binding. The slowrelease in the latter case may be mediated by the immobilized labelledanalyte being surrounded by an environment that promoted binding wherethe mobile phase antagonizes this binding. Essentially this sets up agradient through the matrix housing the adsorbed labelled reactant,until eventually at a particular gradient strength, the adsorbedmaterial will begin to be released. Prior to the release of the labelledreactant, the passage of the mobile phase through the sample adsorptionzone will allow the specific binding of the human antibody specific forthe immobilized ligand in the detection zone without the interferencefrom the labelled reactants with non-specific analytes.

[0089] Translocation of the mobile phase including the immunoreactantsmay be achieved by movement along chambers or channels formed by thebody of the apparatus itself, specific capillary tubing, channels andtroughs or from compartments with flow limiting orifices or movementalong wicks by capillary action. Such wicks may be of synthetic ornatural materials. The choice of material suitable for the translocationmust be such that passive adsorption of the immunoreactants onto theseconducting elements does not occur; the material can be treated toprevent deposition of the immunoreactants on the surface of theconducting elements. If required, the materials used for the conductingelements can either be treated before the device is assembled and/orduring operation of the device. For example, blocking components such asirrelevant proteins, polymers, detergents and other routinely usedblocking reagents common to the practice of immunoassay design shouldnot be present. The capillary elements main purpose is to provide a flowpath for the mobile phase and to connect sections of the apparatus thatcontain immunoreactants or detecting agents that need to be separateprior to conducting the test, and to allow reagents to be added to theimmunoadsorbent at different times. Viscosity modifiers can be used toadjust the rate of translocation together with the flow characteristicsof the conducting elements. Regions of the conducting elements may alsobe modified to perform chemical/immunological interactions with themobile phase and its contents at that particular region of theapparatus. The choice of material for the conducting elements maytherefore be of great importance for the successful operation of theapparatus.

[0090] An important embodiment by which a sequential assay may beperformed by a single addition of mobile phase at the start of theoperation of the apparatus is to have a number of separate conductingelements. In such a system, conducting elements emerge from a singlereservoir which may or may not contain chemically or immunologicallyactive constituents as is required by the particular analyte to bedetected. By allowing a single reservoir to feed a number of conductingelements, this allows different reagents to be applied to the test zoneat various positions and at controlled intervals of time. That is,differentially varying the flow characteristics of each conductingelement will result in different flow rates and hence arrival of variousreagents at different times to the immunoadsorbent. The flow of themobile phase and reactants through a particular conducting element couldalso be modulated by the composition of the conducting element matrixand also by the addition of flow modifiers to each of the conductingelements to change the viscosity. An important embodiment of thisinvention is the circumvention of the requirement of detection agentmodulators. For example, reducing agents may be used to preventpremature chromogen formation when using horseradish peroxidase labelledimmunoreactants.

[0091] The apparatus according to the invention may permit an unlimitednumber of additions of reagents even though they are all initiated froma single reservoir of mobile phase. This will greatly enhance thepotential usefulness of such single step apparatus as the problem ofsequential addition of reagents can be overcome. The multiple conductingelement system circumvents the requirement for discrete wash steps insuch apparatus, allowing the measurement of a wider dynamic range ofanalytes and concentrations, while eliminating the possibility of highdose hook effects which can occur in some non-sequential immunometricassays, that do not have a wash step incorporated to remove unboundanalyte to the solid phase immobilized immunoreactant.

[0092] The multiple conducting elements can be arranged as a sheet intwo dimensions or as a three dimensional structure where more than one“sheet” of conducting elements can be separated by “insulating” layersof material forming a sandwich of conducting elements. For someanalytes, the optimal configuration for the apparatus may be acombination of the two formats especially if more than one analyte is tobe detected at a time. Either way, such a design would allow a complexseries of chemical and immunoreactions to occur separated in time anddistance but ultimately brought together in the correct sequence at thedetection zone to give the required result.

BRIEF DESCRIPTION OF THE DRAWINGS

[0093] An immunoassay analytical test apparatus for allergy diagnosisaccording to the present invention is shown schematically in FIGS. 1 to4 of the accompanying drawings, which are by way of example only. In thedrawings:

[0094]FIG. 1 shows the outward appearance of an immunoassay apparatus;

[0095]FIG. 2 shows an immunoassay apparatus according to the secondembodiment of the present invention, having therein a longer flow pathfor the labelled immunoreactive material relative to the flow path forthe analyte;

[0096]FIG. 3 shows the apparatus according to the first embodiment ofthe present invention, having therein a movable detection zone which isshown in connection with the first flow path; and

[0097]FIG. 4 shows the apparatus of FIG. 3 with the detection zone inconnection with the second flow path.

[0098]FIG. 1 shows the outward appearance of the apparatus (FIG. 1)includes a receptacle for receiving the mobile phase (1), a zone for theaddition of the users sample containing the analyte (2), which may alsobe incorporated in (1), both (1) and (2) being near the surface of theapparatus housing (3). The detection zone (4) is situated within thehousing (3) and may be protected by a transparent window. The detectionzone contains the immunoadsorbent on which the result is observed.

[0099] An important embodiment of the apparatus of FIG. 2 is theconstruction of the conducting elements. In principle, the apparatusconsists of conducting elements of various path lengths and flowcharacteristics. The most rapid and direct path for the mobile phasearises at the point of the reservoir application (1) where the mobilephase is added and leading to the sample application zone (2) situatedon or near the immunoadsorbent. The geometry of the solid phase,together with the materials used to construct the reservoir (1), sink(8), conducting elements (6) and immunoadsorbent (4) are designed topotentiate flow towards the sink (8). The sample application zone leadsto the immobilized reactant on the immunoadsorbent (5) via a filter 9for removing non-IgE components. The labelled antibody zone (7), isfreely mobile within the conducting elements when mobile phase from thereservoir is present and may lead directly to the immunoadsorbent orpass through the sample zone as indicated in FIG. 2. The time taken forthe labelled reactants to reach the immunoadsorbent is longer than forthe sample. This configuration is simple to manufacture from availablematerials and will result in a sequential assay format essentially dueto the difference in arrival times of the sample and labelled antibodyor further reagents at the immobilized immunoreactant zone.

[0100] Referring to FIG. 3, a further embodiment of the presentinvention is represented by a reservoir (1) comprising the mobile phase,a first flow path comprising a sample application zone (2), a filter (9)for removal of non-IgE components and a second flow path comprising alabelled antibody zone (7). There is further provided a movabledetection zone (4), comprising a sink (8). The detection zone (4) isshown in connection with the first flow path.

[0101] Referring to FIG. 4, the apparatus of FIG. 3 is shown with thedetection zone (4) in the second flow path.

[0102] There need not be limitation to two conducting elements asillustrated but there may be a multitude whenever a more complexchemistry of reactants is desired. This arrangement can also be simplyextrapolated to form a three dimensional structure where the conductingelements of various physical properties are stacked one upon anotherprior to adjoining the immunoadsorbent.

EXAMPLES

[0103] The invention will now be illustrated in more detail withreference to the following worked examples.

Example 1

[0104] A piece of Schleicer and Schuell plastic backed nitrocellulose(SSLU) 5 mm×60 mm was prepared as described above. Three lines ofallergen were imprinted within the nitrocellulose 10 mm apartrepresenting Timothy Grass/Cocksfoot Mixed allergens (2 mg/ml), Catallergens (10 mg/ml, Bayer) and Dermatophagoides pteronyssinus extract(20 mg/ml, Smithkline Beecham). A piece of GF/A glass fiber filter paper(Whatman, 5 mm×35 mm) was attached to the proximal end of thenitro-cellulose and a piece of CHR17 (Whatman, 5 mm×40 mm) to the distalend by solvent adhesive to a plastics support, with a 5 mm overlap ontothe nitro-cellulose surface. A 25 μl sample of three serum pools RASTgrade 3 to 4 positive for either grass pollen, cat or mite allergens ora normal goat serum pool were spiked with an equal volume of fresh humannormal donor red blood cells, and added as a 50 μl aliquot to the centreof the GF/A filter. Once absorbed, the free end of the filter was placedin contact with 200 μl of 50 mM Tris buffered saline pH7.4 containing0.5 μg of biotinylated Goat anti-human IgE (Vector Laboratories) andallowed to absorb for 1 hour. The free end of the filter was then placedin contact with 300 μl of the above buffer containing 30 μl of gold (40nm) labelled Goat anti-biotin (British Biocell International) andallowed to absorb. After 30 minutes positive results were observed bythe presence of pink/red lines corresponding to the representativeallergens from each of the serum pools, except for the normal goat serumwhich served as a negative control where no lines were visible for anyof the allergens tested.

Example 2

[0105] A piece of Schleicer and Schuell nitro-cellulose (5×30 mm) wasimpregnated with mite extract as described above. A piece of GF51(Schleicher and Schuell, 5×25 mm) was attached to the nitro-cellulosewith a 5 mm overlap and both components fixed by adhesive to a plasticssupport. A 25 μl aliquot of RAST grade 4 positive serum from a housedust mite positive patient pool was then added to the GF51 to a regionpreviously impregnated with 0.5 μg of biotinylated goat anti-human IgEin 10 μl of Tris buffered saline. A 100 μl aliquot of gold(40 nm)labelled goat anti-biotin in Tris buffer containing 1% w/v bovine serumalbumin was then added dropwise to the GF51 filter. A positive bandcorresponding to reactivity of the serum with the mite extract wasobserved within 30 minutes.

[0106] The reactants, except for the mobile phase, are probably beststored within the apparatus in a solid form to enhance stability, as itis desirable for the apparatus to be stored at ambient conditions.Biological components are notorious for degradation in solution whereasstorage in a dried state or chemically coupled to some matrix conferssome protection to their innate lability. Another problem withbiological materials (especially proteins) is their tendency to adsorbnon-specifically to matrix surfaces such as glass, paper and plastics.These interactions have to be minimized if the reactants are to be ofany use in the apparatus. One method is to pretreat the matrix holdingthe reactants with a non-specific blocking reagent as described above.Another method may be to chemically bind them to the solid phase but toinclude a releasing agent that releases the reactant from the solidphase into the mobile phase. One way to add the reactants is in a liquidform onto the non-adsorbent matrix and then to remove the liquid therebydrying as a film. The reactants could also be added in the form of a gel(such as agarose or gelatin), or simply dried into the matrix with adetergent. The components of the device also contain preservatives tohelp prevent deterioration of the product by microbial organisms and tohelp protect against adverse environmental conditions, for example,temperature light and humidity.

What is claimed is:
 1. Immunoassay analytical test apparatus, whichapparatus comprises: a) a zone for receiving a sample containing ananalyte; b) a zone for receiving a mobile phase, which zone may be thesame as the sample receiving zone, or different thereto; c) detectionmeans for permitting detection of said analyte by immunoreaction; d) afirst flow path for flow of said analyte in said mobile phase from saidsample receiving zone to said detection means; and e) a second flow pathpermitting flow of said mobile phase to said detection means. 2.Apparatus according to claim 1, wherein said second flow path includeslabelled immunoreactive material.
 3. Apparatus according to claim 1,wherein said first flow path includes a material selected form the groupconsisting of: (i) unlabelled immunoreactive material; (ii) haptenlabelled immunoreactive material; (iii) unlabelled capture material;(iv) hapten labelled capture material; and (v) detector labelledmaterial; said material being upstream of said sample receiving zone. 4.Apparatus according to claim 1, wherein said first flow path includes amaterial selected form the group consisting of: (i) unlabelledimmunoreactive material; (ii) hapten labelled immunoreactive material;(iii) unlabelled capture material; (iv) hapten labelled capturematerial; and (v) detector labelled material; said material beingdownstream of said sample receiving zone.
 5. Apparatus according toclaim 1, wherein said first flow path includes a material selected formthe group consisting of: (i) unlabelled immunoreactive material; (ii)hapten labelled immunoreactive material; (iii) unlabelled capturematerial; (iv) hapten labelled capture material; and (v) detectorlabelled material; said material being upstream and downstream of saidsample receiving zone.
 6. Apparatus according to claim 1, wherein saidflow paths are such that said mobile phase is allowed to traverse morethan one path simultaneously, said first flow path being continuous fromsaid mobile phase receiving zone to said detection means, said secondflow path being continuous from said mobile phase receiving zone to saiddetection means.
 7. Apparatus according to claim 1, wherein saiddetection means is movable from a first position in communication withsaid first flow path to a second position in communication with saidsecond flow path.
 8. Apparatus according to claim 7, wherein saiddetection means is movable from a first position in contact with saidfirst flow path to a second position in contact with said second flowpath.
 9. Apparatus according to claim 7, wherein said detection means ismovable to be in communication with said first and second flow paths insequence.
 10. Apparatus according to claim 7, wherein said detectionmeans is movable to be in contact with said first and second flow pathsin sequence.
 11. Apparatus according to claim 1, wherein said first flowpath potentiates flow towards said detection means.
 12. Apparatusaccording to claim 11, wherein said first flow path potentiates flowtowards said detection means by a capillary action.
 13. Apparatusaccording to claim 1, wherein said second flow path potentiates flowtowards said detection means.
 14. Apparatus according to claim 13,wherein said second flow path potentiates flow towards said detectionmeans by a capillary action.
 15. Apparatus according to claim 1, whereinsaid first flow path is selected from the group consisting of: a)elongate sheet material; b) elongate strip material; and c) materialabsorbent to said mobile phase.
 16. Apparatus according to claim 15,wherein said elongate sheet or strip comprises a portion contactablewith said first flow path.
 17. Apparatus according to claim 1, whereinsaid second flow path is defined by a material selected from the groupconsisting of: a) elongate sheet material; a) elongate strip material;and b) material absorbent to said mobile phase.
 18. Apparatus accordingto claim 17, wherein said elongate sheet or strip comprises a portioncontactable with said second flow path.
 19. Apparatus according to claim1, wherein said analyte is allergen specific IgE.
 20. Apparatusaccording to claim I, wherein said apparatus further comprises a sinkfor collection of excess material exiting the detection means. 21.Apparatus according to claim 1, wherein said detection means is manuallymovable from said first pathway to said second pathway.
 22. Apparatusaccording to claim 19, wherein said first flow path comprises a matrixfor the removal of non-IgE components.
 23. Apparatus according to claim22, wherein said matrix is provided between said sample receiving zoneand said detection means.
 24. Apparatus according to claim 22, whereinsaid matrix is provided in said sample receiving zone.
 25. Apparatusaccording to claim 1, wherein said first flow path comprises a filterarranged to separate components of said sample, said filter selectedfrom a group consisting of: i) a blood filter arranged to permit plasmato pass whilst capturing other blood constituents; and ii) a matrix forthe removal of material other than said analyte.
 26. Apparatus accordingto claim 25, wherein said filter is provided between said samplereceiving zone and said detection means.
 27. Apparatus according toclaim 25, wherein said filter is provided in said sample receiving zone.28. Apparatus according to claim 1, wherein material comprising saidfirst flow path enables transport of said sample along said first flowpath.
 29. Apparatus according to claim 1, wherein material comprisingsaid first flow path enables transport of at least a constituent of saidsample along said first flow path.
 30. Apparatus according to claim 1,which includes a store of mobile phase arranged to be released upstreamof said sample.
 31. Apparatus according to claim 30, wherein said storeis contactable with at least a part of said first flow path. 32.Apparatus according to claim 30, wherein said store is contactable withat least a part of said first flow path and at least a part of saidsecond flow path.
 33. Apparatus according to claim 1, further comprisinga separate container which contains said mobile phase, wherein saidmobile phase may be released into said mobile phase receiving zone. 34.Apparatus according to claim 1, wherein said second flow path isconvoluted and includes a zone containing labelled immunoreactivematerial capable of reacting with said analyte to produce labelledanalyte, such that unlabelled analyte may reach and becomes immobilizedin said detection zone via said first path before said labelledimmunoreactive material reaches said detection zone via said second pathwhereby said labelled immunoreactive material can react with immobilizedanalyte.
 35. Apparatus according to claim 34, wherein said second flowpath comprises at least a part of said first flow path.
 36. Apparatusaccording to claim 34, wherein said second flow path comprisessubstantially the entirety of said first flow path.
 37. Apparatusaccording to claim I which comprises a plurality of flow paths, whereinsaid plurality of flow paths are stacked.
 38. An immunoassay analyticaltest method utilizing apparatus according to claim
 1. 39. Immunoassayanalytical test apparatus, which apparatus comprises: a) a zone forreceiving a sample containing an analyte; b) a zone for receiving amobile phase, which zone may be the same as the sample receiving zone,or different thereto; c) detection means for permitting detection ofsaid analyte by immunoreaction; d) a first flow path for flow of saidanalyte in said mobile phase from said sample receiving zone to saiddetection means; and a) a second flow path permitting flow of saidmobile phase to said detection means; wherein said detection means ismovable from a first position in communication with said first flow pathto a second position in communication with said second flow path. 40.Apparatus according to claim 39, wherein said detection means is movablefrom a first position in contact with said first flow path to a secondposition in contact with said second flow path.
 41. Apparatus accordingto claim 39, wherein said detection means is movable to be incommunication with said first and second flow paths in sequence. 42.Apparatus according to claim 39, wherein said detection means is movableto be in contact with said first and second flow paths in sequence. 43.Apparatus according to claim 39, wherein said second flow path includeslabelled immunoactive material.
 44. Apparatus according to claim 39,wherein said first flow path includes a material selected form the groupconsisting of: (i) unlabelled immunoreactive material; (ii) haptenlabelled immunoreactive material; (iii) unlabelled capture material;(iv) hapten labelled capture material; and (v) detector labelledmaterial; said material being upstream of said sample receiving zone.45. Apparatus according to claim 39, wherein said first flow pathincludes a material selected form the group consisting of: (i)unlabelled immunoreactive material; (ii) hapten labelled immunoreactivematerial; (iii) unlabelled capture material; (iv) hapten labelledcapture material; and (v) detector labelled material; said materialbeing downstream of said sample receiving zone.
 46. Apparatus accordingto claim 39, wherein said first flow path includes a material selectedform the group consisting of: (i) unlabelled immunoreactive material;(ii) hapten labelled immunoreactive material; (iii) unlabelled capturematerial; (iv) hapten labelled capture material; and (v) detectorlabelled material; said material being upstream and downstream of saidsample receiving zone.
 47. Apparatus according to claim 39, wherein saidfirst flow path potentiates flow towards said detection means. 48.Apparatus according to claim 47, wherein said first flow pathpotentiates flow towards said detection means by a capillary action. 49.Apparatus according to claim 39, wherein said second flow pathpotentiates flow towards said detection means.
 50. Apparatus accordingto claim 49, wherein said second flow path potentiates flow towards saiddetection means by a capillary action.
 51. Apparatus according to claim39, wherein said first flow path is selected from the group consistingof: a) elongate sheet material; b) elongate strip material; and c)material absorbent to said mobile phase.
 52. Apparatus according toclaim 51, wherein said elongate sheet or strip comprises a portioncontactable with said first flow path.
 53. Apparatus according to claim39, wherein said second flow path is defined by a material selected fromthe group consisting of: a) elongate sheet material; a) elongate stripmaterial; and b) material absorbent to said mobile phase.
 54. Apparatusaccording to claim 53, wherein said elongate sheet or strip comprises aportion contactable with said second flow path.
 55. Apparatus accordingto claim 39, wherein said analyte is allergen specific IgE. 56.Apparatus according to claim 39, wherein said apparatus furthercomprises a sink for collection of excess material exiting the detectionmeans.
 57. Apparatus according to claim 39, wherein said detection meansis manually movable from said first pathway to said second pathway. 58.Apparatus according to claim 55, wherein said first flow path comprisesa matrix for the removal of non-IgE components.
 59. Apparatus accordingto claim 58, wherein said matrix is provided between said samplereceiving zone and said detection means.
 60. Apparatus according toclaim 58, wherein said matrix is provided in said sample receiving zone.61. Apparatus according to claim 39, wherein said first flow pathcomprises a filter arranged to separate components of said sample, saidfilter selected from a group consisting of: i) a blood filter arrangedto permit plasma to pass whilst capturing other blood constituents; andii) a matrix for the removal of material other than said analyte. 62.Apparatus according to claim 61, wherein said filter is provided betweensaid sample receiving zone and said detection means.
 63. Apparatusaccording to claim 61, wherein said filter is provided in said samplereceiving zone.
 64. Apparatus according to claim 39, wherein materialcomprising said first flow path enables transport of said sample alongsaid first flow path.
 65. Apparatus according to claim 39, whereinmaterial comprising said first flow path enables transport of at least aconstituent of said sample along said first flow path.
 66. Apparatusaccording to claim 39, which includes a store of mobile phase arrangedto be released upstream of said sample.
 67. Apparatus according to claim66, wherein said store is contactable with at least a part of said firstflow path.
 68. Apparatus according to claim 66, wherein said store iscontactable with at least a part of said first flow path and at least apart of said second flow path.
 69. Apparatus according to claim 39,further comprising a separate container which contains said mobilephase, wherein said mobile phase may be released into said mobile phasereceiving zone.
 70. Apparatus according to claim 39, wherein said secondflow path is convoluted and includes a zone containing labelledimmunoreactive material capable of reacting with said analyte to producelabelled analyte, such that unlabelled analyte may reach and becomesimmobilized in said detection zone via said first path before saidlabelled immunoreactive material reaches said detection zone via saidsecond path whereby said labelled immunoreactive material can react withimmobilized analyte.
 71. Apparatus according to claim 70, wherein saidsecond flow path comprises at least a part of said first flow path. 72.Apparatus according to claim 70, wherein said second flow path comprisessubstantially the entirety of said first flow path.
 73. Apparatusaccording to claim 39 which comprises a plurality of flow paths, whereinsaid plurality of flow paths are stacked.
 74. An immunoassay analyticaltest method utilizing apparatus according to claim 39.